[show abstract][hide abstract] ABSTRACT: The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products.
[show abstract][hide abstract] ABSTRACT: Gap junctions, composed of connexin (Cx) subunits, are channels that allow intercellular communication between adjacent cells and are thought to play a key role in the regulation of cell proliferation and differentiation. The Cx expression pattern and formation of gap junctions in human fetal hair follicles has yet to be clarified, including the prominent follicular bulge region that is believed to be a site rich in stem cells.
To study the expression of two major Cxs, Cx26 and Cx43, in developing hair follicles in skin samples from a series of human fetuses of estimated gestational age (EGA) 88-163 days, and to determine quantitatively the presence of gap junctions.
We used immunofluorescence labelling to investigate the sequential expression pattern of Cx26 and Cx43 in developing human hair follicles. Gap junction formation was observed by electron microscopy and the numbers of gap junctions were analysed quantitatively. Results Both Cx26 and Cx43 expression were observed at 88 days' EGA in the inner part of the hair peg. At 135 days' EGA, Cx26 was expressed in the outer root sheath (ORS) and the inner root sheath (IRS), while Cx43 was expressed chiefly in the IRS, hair matrix and sebaceous glands. At 163 days' EGA, Cx26 expression was most intense in the outermost layer of the ORS, in contrast to Cx43 expression which was in the inner part of the ORS. In the bulge region, only Cx43 was expressed in a subset of cells in the bulge. Ultrastructurally, gap junctions were observed at 102 days' EGA in the hair peg, and the number of gap junctions increased as the hair follicle matured. Gap junctions were also observed between the bulge cells in considerable numbers.
The changing expression patterns of Cx26 and Cx43 and the increasing gap junction numbers suggest a close association of Cx expression and gap junction formation with hair follicle morphogenesis. In addition, the present ultrastructural observations demonstrate that considerable numbers of the bulge cells, a putative site rich in hair follicle stem cells, form gap junctions during human hair follicle development.
British Journal of Dermatology 04/2004; 150(3):429-34. · 3.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Peptidylarginine deiminases are widely distributed, calcium-ion-dependent enzymes that convert arginine residues of proteins into citrulline residues. This reaction, deimination, is thought to be an important event during the final stage of epidermal differentiation, possibly associated with integration and disintegration of keratin filaments. To elucidate the possible roles of protein deimination during human epidermal development we investigated localization of deiminated proteins using anti-citrulline peptide antibody, which preferentially recognizes citrulline residues in the V subdomains of keratin 1, and anti-chemically modified citrulline antibody, which enables detection of citrulline residues independent of amino acid sequences. Anti-chemically modified citrulline antibody, but not anti-citrulline peptide antibody stained the periderm in two-layered epidermis of 49 d and 57 d estimated gestational age. In the stratified epidermis of 88 d, 96 d, and 108 d estimated gestational age fetal skin, anti-citrulline peptide antibody and anti-chemically modified citrulline antibody staining was seen in the periderm and intermediate cell layers. After periderm cells regressed and keratinization began in the interfollicular epidermis, anti-citrulline peptide antibody and anti-chemically modified citrulline antibody were restricted to the cornified cell layers of the interfollicular epidermis, similar to the distribution patterns of that in adult epidermis. Postembedding immunoelectron microscopy showed anti-citrulline peptide antibody immunogold labeling over the cytoplasmic intermediate filament network in the periderm and the intermediate cell layers. These results demonstrate an orderly formation of deiminated proteins in different layers of embryonic epidermis and suggest important roles for peptidylarginine deiminases in human epidermal morphogenesis.
Journal of Investigative Dermatology 06/2003; 120(5):817-22. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Erythrokeratoderma variabilis, characterized by migrating erythema and fixed keratotic plaques, is a rare congenital disorder which has recently been connected with connexin (Cx)30.3 or Cx31 gene mutations. We present a 9-month-old Japanese girl who exhibited the typical clinical features of the disease, but carried no Cx30.3 or Cx31 gene mutations. Histopathologically, regular acanthosis with hyperkeratosis and hypergranulosis was observed in her lesional skin. Upregulation of involucrin and loricrin expression, and a weak expression of Cx26 was immunohistochemically observed in the upper spinous and granular layers. Electron microscopy revealed no abnormality in the keratin filaments, cornified cell envelope or gap junctions. Direct sequencing revealed no pathogenetic mutations in the Cx26, Cx30.3, Cx31 or Cx31.1 genes in this patient. The results indicate that erythrokeratoderma variabilis is pathologically heterogeneous, and that abnormalities in keratinization other than Cx30.3 and 31 gene mutations may underlie some forms of this disease.
[show abstract][hide abstract] ABSTRACT: Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.
Journal of Histochemistry and Cytochemistry 12/2002; 50(11):1493-500. · 2.26 Impact Factor